Growing microorganisms in order to detect, enumerate and identify viable cells—bacteria, fungi, actinomycetes—is one of the most widely used procedures in microbiology. The ability to form a colony on or in an appropriate nutrient media is recognized as the most reliable criteria for cell viability. Formation of a colony requires different biochemical reactions and processes like respiration, transportation, synthesis and decomposing of different proteins, carbohydrates, lipids, enzymes, nucleic acids and many other substances, and creation of inner structures in order to finally reach cell reproduction and creation of a colony. The detection of live cells cannot be fully substituted by the simple detection of enzymes (enzymatic methods of detection), purified DNA or RNA (PCR methods), antigen-antibody interactions (Enzyme Immunological Analysis, ELISA, Immunoprecipitation, Immunofluorescence, Phagodiagnostics and others), fatty acids analysis (Chromatography), FTIR spectroscopy or other methods. This is due to the presence of specific molecules or even some working systems of the cell, which can be found in dead cells, cells under deadly stress, or cells without access to some required substances that are therefore restricted in growth potential.
Growth of microorganisms takes from hours to several days, or even weeks to form well visible colonies or a visible suspension of cells. The growth occurs either on artificial or natural nutrient media in solid or liquid form. There is a multitude of different media with selective criteria required for growth and total count of groups or species of microorganisms. These include media for growth, detection and enumeration of total number of bacteria (Tryptic Soy Agar and other), fungi, molds (Sabouraud Dextrose Agar, Potato Dextrose Agar and other), selective media for group of microorganisms like Gram-negative bacteria (MacConkey Agar, Levine EMB Agar and other), Lactobacillius (Lactobacillii MRS Agar) or Salmonella (SS Agar), certain microbes like E. coli O:157 (Sorbitol MacConkey Agar), Vibrio chelerae (TCBS Agar), Campylobacter (Triple Sugar Iron Agar) and many others. Growing periods on nutrient media is the most time consuming process in modern microbiological diagnostics. Reducing the time between the inoculation and detection of microorganisms is very important for rapid decisions in quality and process control in a multitude of industries including food, biotechnological, pharmaceutical, water treating industry, and also in medical microbiological diagnostics, environmental and biodefense control and monitoring, and scientific research. Thus, for example, significantly decreasing the time of microbiological analysis together with saving the high level of reliability (i.e., detection by growth—forming of colonies) is very important for modern medical diagnostics for early diagnostics of humans or animals infections, epidemiology, and detection of antibiotic resistant microbes and so on. Rapid analysis (analysis significantly faster than regular growth and further analysis) is also needed in food, pharmaceutical, and biotechnological industries for control and prevention of contamination in food, drugs, and medical devices and for environmental monitoring.
The term “colony” or “micro colony” in microbiology means a group of cells appearing from one single cell and consisting only from descendants of that cell. A colony or micro colony can have different shapes: semi-sphere if grown on the surface of solid agar, oval, cone or “star” if grown in solid or semi-solid agar, a dense or diffuse cloud if grown on semi-liquid or liquid media, or flat fibers growing from one center (fungi, actinomycetes, or some bacterial micro colonies).
There are several different methods and instruments employed to enhance colony visibility. The addition of special, non-toxic substances (artificial chromogenic or fluorogenic substrates) to solid nutrient media changes the color of the micro-colonies or makes them fluorescent. Some microorganisms like E. coli O:157, Staphilococcus aureus, and Salmonela grows on “Chromagars,” (Hardy Diagnostics, Inc. CA, CROMagar Company, France) a solid or semi-solid nutrient media that specifically changes the color of investigated microorganisms because of artificial substrates for unique enzymes added to media. These substrates are non-toxic to the cells and allow normal growth. Toxic artificial substrates such as Tetrazolium salts, Fluorescein diacetate and other substances can not be used because stops cells growth. Chromagars' time of incubation is typically 24-120 hours, which is not considered rapid.
Detection and enumeration of colonies are done visually with a naked eye or with magnifying devices. Visual detection and enumeration using magnifying devices requires relatively big colonies; from hundreds of microns to millimeters in diameter. Microscopy helps to find micro-colonies that are smaller in size, however, these colonies must be at least tens of microns in size, contain at least several hundred cells, and require at least 10-12 hours of incubation. Additional coloration of these micro-colonies is difficult because cells spread/wash away on the surface when dye solution applied. It is also difficult to find them on a big surface of plate.
Detection of microorganisms could also be achieved by dividing a sample into many discrete zones, adding liquid nutrient media, incubating from many hours to days, adding indicator substance or counting turbid zones, and calculating concentration (U.S. Pat. No. 5,716,798). This method gives a reduction of growth of only 20-40% because employed relatively large volumes of discrete zones, consisting of large wells on the side of a special flask for growth. A 90% of zone volume related to nutrient media and therefore analyzing sample could be only several milliliters. Therefore, this method is used only for the detection of microbes in human blood because blood samples are very small and their contamination is in a range from single cells to several hundred cells per milliliter. The same idea used in SIMPLATE™ device (Biocontrol Systems, WA, USA)—device for growth of microorganisms in liquid media divided on several tens smaller volumes (U.S. Pat. Nos. 5,518,892, 5,620,895 and 5,753,456). The time of incubation in the SIMPLATE™ device is 24 hours.
The modern microbiology employs some methods to shorten time for microorganism growth and to improve the visibility of colonies, for example, employing optimal growth nutrient media, adding chemical matter in the nutrient media, or employing optical instruments or devices. However, there are no methods utilizing the shape of the colony during its growth in order to enhance its optical density (light absorbance) or fluoresence. The shape of a regular micro-colony is usually semi-sphere. See FIG. 1A. Changing a colony's shape from a regular semi-sphere with a large volume and a large amount of cells to a thin cylinder shape with a small volume and a small amount of cells will strongly reduce the time between inoculation and colony counting. Smaller amounts of cells need a shorter time for their production. The usage of chemicals producing color or fluorescence and optical instruments together with detection of cylindrical colonies would improve visibility and reduce the time required for analysis.
Therefore, there remains a need in the art for new methods and apparatus of early microorganism detection that have quicker microorganism recognition time than existing methods and apparatuses.